PROJECT SUMMARY: While tremendous progress has been made to understand the roles of epithelial pro- genitors in tissue repair and fibrotic mechanisms, little is known about the origin, nature of interaction and gene regulatory programs of fibroblast progenitors. Pulmonary research has been challenged by the remarkable complexity and diversity of fibroblast populations and mesenchymal stem cell populations. Our long-term goal is to understand the complexity of fibroblast populations and to identify their regulatory role on the epithelium during alveolarization and epithelial repair. Our primary objective is to identify key regulators of fibroblast dif- ferentiation in the lung. The present application is based on our preliminary data that identifies GATA6 as a key transcription factor in matrix iReFs, which are indispensable for alveolarization. Our central hypothesis is that GATA6 regulates gene expression in iReFs that sub sequentially induce matrix iReF specification and para- crine interactions with AECs during development and repair. The rationale for the proposed research is that understanding the regulation of fibroblast subpopulations, and their distinct roles in epithelial-mesenchymal cell interactions, will provide new answers for fundamental questions regarding lung homeostasis and repair. This hypothesis will be tested with three specific aims: 1) that matrix iReFs instruct AEC1 differentiation. 2) that GATA6 transcriptionally regulates a set of matrix fibroblast signature genes and 3) that H3K27me silencing marks regulate GATA6 and matrix gene expression in aged iReFs. In the first aim, alveolosphere cultures will be used to interrogate the epithelial-mesenchymal crosstalk between specific iRef subpopulations and wild type alveolar epithelial cells. In the second aim, gain and loss of function studies of GATA6 will be used to es- tablish a functional link between fibroblast phenotype and alveolarization. In the third aim, H3K27me3 histone marks will be assessed by ChIP-PCR analysis. In vivo and in vitro studies using EZH2 inhibitors will determine a role of EZH2 in suppressing matrix fibroblast signature genes. These studies will provide a better under- standing of the impact of fibroblast diversity on epithelial-mesenchymal crosstalk during alveolarization and alveolar regeneration. This contribution will be significant because it will advance the knowledge about genes and pathways involved in alveolar regeneration, give new insights into the cause of decreased regrowth with age and provide the basis for new therapies to overcome current limitations of regeneration in the human lung. Our understanding of the role of lung fibroblasts in lung injury and regeneration lags far behind our understand- ing of the role of epithelial cells. New information about interstitial lung fibroblasts will be invaluable for defining mechanisms of development, normal lung repair, and lung disease.